Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

Induction of in vitro androgenesis in anther and isolated microspore culture of different spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) genotypes

This is the first report on isolated microspore culture—derived spelt wheat. The efficiency of anther- and isolated microspore was compared using four genotypes (‘Franckenkorn’, ‘GK Fehér’, ‘Mv Martongold’, ‘Oberkulmer Rotkorn’). In anther culture, genotype dependency was observed, and cold pre-treatment enhanced the efficiency of the method. In isolated microspore culture, the ovary co-culture supported the development of embryo-like structures. The presence of growth regulators (0.5 mg/l 2,4-D and 0.5 mg/l kinetin) were not essential for the induction of androgenesis, but these increased the production of embryo-like structures, green and albino plantlets. The low plant regeneration rate and high number of albinos hinder the practical application of isolated microspore culture while anther culture was efficient for in vitro green plantlets production in spelt wheat. The mean of green plantlets production was 41.45/100 anthers (from 20.93 to 83.07 depending on genotype). The phenomenon of albinism was mitigated in anther culture (3.48 albinos/100 anthers). Altogether, 1720 anther culture—derived green plantlets were produced from the four genotypes.     

Effects of exogenous application of polyamines on wheat anther cultures

Androgenesis of wheat genotypes was evaluated by pretreating anthers or embryo-like structures (ELS) with polyamines. Anthers of the genotype DH were pretreated with different concentrations of putrescine, spermidine, and spermine for 1, 3, and 6 h, and those of drought-tolerant International Center for Agricultural Research in the Dry Areas (ICARDA) wheat accessions were treated for 1 and 3 h. ELS of two genotypes were also treated for 30 and 60 min with the same polyamines and evaluated for green plant regeneration. The pretreatment of anthers with polyamines enhanced the development of ELS in all genotypes. The formation of ELS varied significantly with genotype. Pretreated anthers showed that four treatments improved significantly green plant regeneration with the genotype ICR 17. However, two treatments (1 mM putrescine or spermine for 1 h) significantly improved green plant regeneration per 100 ELS of only two ICARDA genotypes. ELS treated with polyamines for 30 min were greener and formed more adventitious roots. The chloroplasts of these greener ELS examined with a transmission electron microscope had agranal to grana thylakoids, while those of the control had plastids with mostly starch globules. Although exogenous application of polyamines to anthers improved the production of ELS and green plants, the effects of putrescine, spermidine, and spermine was dependent on genotype and the duration of pretreatment of anthers with the polyamines.     

Comparison of media for their aptitude in wheat anther culture

Different media were evaluated with anthers of five spring wheat (Triticum aestivum L.) genotypes for their ability to produce embryos and green plants in anther culture. Our first experiment showed that the addition of a combination of 19 amino acids significantly increased the number of embryos and green plants obtained. The mean number of green plants per 100 anthers for the two genotypes in this experiment, HY320 and B723, went from 28.2 without amino acids in the medium, to 46.7 with addition of amino acids. Our second experiment with the genotypes HY320, Wim and Laval-19 showed that liquid medium with Ficoll is more efficient for anther culture (9.9 green plants/100 anthers) than solid (0 green plants), gelationous media (2.5 green plants/100 anthers) or liquid medium with Membrane Rafts (0 green plants; Hoechst Celanese Corp.). Our third experiment revealed that the effect of replacement of sucrose by maltose varied with the genotype of the donor plant. Maltose partially inhibited the androgenesis of three responsive genotypes, HY320, Wim and Reliance (40.3 green plants/100 anthers instead of 43.9 with sucrose), while maltose significantly increased the androgenesis of the recalcitrant genotype Laval-19 (10.8 green plants/100 anthers instead of 5.4 with sucrose). An amino acid x maltose interaction was also observed. Amino acids without maltose increased androgenesis, but the addition of maltose to the amino acid-enriched medium eliminated this positive effect of the amino acids.     

Influence of medium and cold pretreatment on androgenetic response in Lolium perenne L.

Summary Genotypes of Lolium perenne L. with different androgenetic responses were used to test effects of induction medium composition. The media tested were potato II (pII), 190-2, and modified Linsmaier and Skoog media, LS-1, LS-2, and LS-3. The effect of different gelling agents, activated charcoal in a double layer design, and casein hydrolysate were also studied. From 36,696 anthers, 25,906 embryo-like structures, 1,959 albino and 173 green plants were generated. Significant differences were found between media, genotypes and medium-genotype interactions studied. All three media commonly used, pII, 190-2, and LS-3, were equivalent in production of green plants. Cold pretreatment of the anthers (4°C) significantly increased the number of embryo-like structures, the number and proportion of albino plants produced, but not the production of green plants.Abbreviations ELS embryo-like structures - ALB albino plants - ANT anthers - GRP green plants - DH doubled haploid plants - AC activated charcoal - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog (1965) basal medium - MS Murashige and Skoog (1962) - PL plants - pII potato II induction medium - DL double layer     

Genotypic variation of quantitative trait loci controlling in vitro androgenesis in maize.

A quantitative trait loci (QTL) analysis for androgenetic capability has been conducted on three different crosses in maize, including very high and nonresponding lines for androgenesis. The doubled haploid lines derived by anther culture from the crosses DH5 x DH7, A188 x DH7, and R6 x DH99 showed a range of 0-70%, 0-40%, and 0-50% androgenetic responding anthers, respectively. The genotypic heritability of means for this trait is close to 0.90 for A188 x DH7 and 0.78 for R6 x DH99. The QTL analysis involved in each population the mapping of more than 100 loci covering a large part of the genome with reasonably spaced markers averaging 12 cM. Different measurements describing the androgenetic process were studied: AC, percentage of responding anthers; ELS, number of androgenetic embryos produced per 100 plated anthers; PLE, number of plantlets regenerated per 100 embryos; PLA, number of plantlets per 100 plated anthers. In each cross, three to four QTLs were found for AC, explaining 30-40% of the phenotypic variation. The QTL detected for PLA was also strong QTL for AC or ELS. This agrees with the observation that these last two traits are good predictors for final plantlet yield. The QTLs found were specific, although the same line DH7 was used in two crosses and DH99 derived from DH5 and DH7 in the third cross. These results suggest that the transfer of the androgenetic capabilities in elite germplasm will still involve a phenotypic evaluation of the androgenetic performances. A backcross-assisted selection based only on the genotype at the QTL is probably possible but only within the crosses used for this QTL analysis.     

The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum)

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to –2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with –0.9 to –1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.     

Ovary co-culture improves embryo and green plant production in anther culture of Australian spring wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

A simple anther culture protocol for Australian spring wheat cultivars was developed using ovary co-culture. The inclusion of ovaries in the induction medium significantly increased the production of embryo-like structures (ELS), green and albino plants in two spring wheat cultivars tested. When five ovaries were added to the induction medium, the mean number of ELS per spike increased from 7.6 to 50.1 and green plants per spike increased from 0.6 to 8.9. The addition of 10 ovaries, however, did not further increase the production of ELS or green plants. The growth regulator combination of 2,4-D and KIN was compared with IAA and BA. There were no significant differences in the numbers of ELS or green plants although significantly fewer albino plants were produced with IAA and BA. Eight additional cultivars were screened using the protocol with either 5 or 10 ovaries in the induction medium. Green plants were obtained from nine varieties at frequencies ranging from 0.3 to 33.0 green plants per spike. Regenerant plants at maturity exhibited chromosome fertility rates in different cultivars ranging from 15% to 100%.     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Functional properties of ficoll and their influence on anther culture responses of wheat

The effects of ficoll in liquid culture media have been contradictory in previous reports. The objective of this study was to determine the functional properties of ficoll in potato 4 (P4) liquid induction medium and their influence on anther culture responses of wheat. Ficoll addition significantly (p0.01) reduced callus production from the anthers of spring wheat cv. Pavon 76. The reduction was directly related to the concentration of ficoll added within the range of 50 to 200 g l^-1 medium. Although the addition of ficoll significantly (p0.01) increased the percentage of regenerable calli and the ratio of green vs. albino plants, the final yield of green plants per 100 anthers was significantly lower. Consistent results also were obtained with four other spring wheat genotypes (Chris, Butte 86, WA 6916, and Edwall). Ficoll concentration affected the density, viscosity, and osmolality of the liquid media. The higher medium density caused by ficoll addition increased the percentage of floating calli, as well as the percentage of regenerable calli and the ratio of green vs. albino plants. However, the increased medium viscosity by ficoll addition significantly (p0.01) reduced callus production. Ficoll addition also increased medium osmolality, which affected callus production by interacting with the sugar concentration of the induction media. Using response functions, the estimated maltose concentration for maximum callus production was 105 g l^-1 for the standard P4 media, compared with 68 g l^-1 for the ficoll-containing P4 media. These results clearly demonstrate that ficoll addition to the liquid P4 induction medium containing high sucrose concentration (90 g l^-1) is deleterious to the maximum production of green plants from wheat anther culture.     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

Effect of genome, induction medium and temperature pretreatment on green plant production in durum ( Triticum turgidum var. durum ) x bread wheat ( Triticum aestivum L. em Thell) crosses

The recalcitrancy of durum wheat (Triticum turgidum var. durum) to anther culture, was attempted to be overcome by transferring the responsible genes form bread wheat B-genome to the respective on durum wheat, determining an appropriate induction medium and clarifying the necessity of cold pretreatment. For this, three durum wheat cultivars were crossed to two bread wheat (Triticum aestivum L. em Thell) cultivars. The resulting F₁ plants and their original cultivars were grown in the field and anthers at the appropriate microspore stage were cultured on potato-2 and W₁₄ media with and without low temperature pretreatment. No green plants were produced from the parental durum wheat cultivars. In contrast, green plants were produced from the F₁ plants. The best results in three of the four F₁ hybrids were recorded when potato-2 was used as induction medium. A more variable response of the examined genotypes was noticed with respect to temperature pretreatment. Regarding green plant production, a negative effect of cold pretreatment was observed in two of the F₁ hybrids when they were cultured on potato-2. Chromosome counts on root tips from the resulting green plants revealed that they all carried D-genome chromosomes. The last observation could suggest that D-genome chromosomes are necessary for anther culture response in wheat. Yet, the production of one green plant with 15 chromosomes may indicate that the development of extracted durum genotypes from bread wheat genotypes with good response to in vitro anther culture might be possible. Further work however, is needed for this to be verified.     

Float culture of wheat anthers

Summary Experiments on wheat anther culture in liquid media either synthetic or with potato extract show that it is possible to obtain as many embryos as when using solid potato extract medium. In liquid media young embryos or 14-day old induced anthers can differentiate green plants for regeneration. Glutamine is effective in culturing anthers and can replace potato extract in the medium.     

Osmotic potential of media affecting green plant percentage in wheat anther culture

The percentage of green plants in anther culture is known to be controlled by the genetics of anther donor materials. The objective of this study was to determine whether components in the culture media also would have a significant influence on the percentage of green plants from wheat anther culture. Anthers of a spring wheat cultivar, Pavon 76, were cultured on potato 4 (P4) induction media with various modifications. Addition of 200 g/l ficoll to the liquid P4 medium significantly increased the percentage of green plants even though the final yield of green plants per 100 anthers was lower than the liquid medium. A higher concentration of maltose (135 g/l) produced significantly higher percentage of green plants than the medium containing 90 g/l maltose or sucrose. These results demonstrate culture medium effects on albinism, indicating that the percentage of green plants in wheat anther culture can be increased by optimizing medium osmotic potential.     

Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants

Double haploids (DH), obtained during androgenesis in vitro or by genome diploidisation in regenerated haploids, are one type of basic materials used in triticale breeding programmes. The aim of this study was to improve DH production by a combination of colchicine treatment methods on a sample of five winter and five spring triticale hybrids. Colchicine was applied in vitro either in the C17 medium to induce embryo-like structures (ELS) or in the 190-2 medium for green plant (GP) development. Regenerants which remained haploid were immersed in a colchicine solution either when placed on the medium prior to transferring to soil or when growing in pots, followed by the application or absence of cooling. Colchicine treatment during anther culture affected neither ELS nor GP development, but significantly increased the number of DH plants in comparison to spontaneous chromosome doubling. The highest efficiency was recorded when colchicine was applied in the induction medium (55%) versus the regeneration medium (44.5%) or no colchicine treatment (30%). The effectiveness of chromosome duplication in haploid plants ranged from 32 to 64.5% and it was the highest for the treatment on the medium followed by cooling. Individual hybrids differed regarding their capability of regeneration and chromosome doubling, which were consistent only to a low or moderate extent. However, taken together, winter and spring hybrids did not differ significantly. Combined colchicine application resulted in a high yield of DH production, 82.6% for all triticale hybrids, and can provide a considerable number of fertile DH lines for triticale breeding programmes.     

Screening South African wheat germplasm for androgenic competence

Microspore and anther cultures provide an opportunity to create haploid and doubled haploid plants within a single season, thereby reducing the time and cost of cultivar development. Microspore and anther culture has been widely used and incorporated into wheat breeding programs in many countries, but little is known about the effectiveness of these techniques on South African germplasm. By using two responsive genotypes, isolated microspore culture was shown as more effective at revealing androgenic competence, and was used to evaluate the response of four South African inbred lines and two hybrids. Inbred lines A and B were highly responsive (336 and 207 embryo-like structures [ELS] per 100 anthers, respectively), line D was slightly responsive (5.1 ELS per 100 anthers) while line C was recalcitrant. The hybrid A × C was highly responsive (274 ELS per 100 anthers), and B × D did not respond at all. Green plant regeneration in a local genotype was very low (1% for line B) compared to that of foreign genotype (17% for Pavon 76). Similarly to other wheat genotypes grown around the world, the responsiveness of the South African varieties is also very variable. Thus, more efforts are needed so that isolated microspore culture can become a general tool in breeding programs.     

Barley anther culture: effects of annual cycle and spike position on microspore embryogenesis and albinism

The effect of donor plants annual cycle and anther/spike position on the production of microspore-derived plants and albinism were studied. We used the winter cv. Igri and the spring cv. Cork, known to respond similarly in anther culture but to produce 78% and 2% of green plants, respectively. In both cvs. the number of microspore-derived plants was significantly higher when the anthers were collected from January to July than from August to December. However, during this period the proportion of albino plants was not altered. Conversely, the anther response decreased from 76.6 to 31.5% in Igri and from 58.8 to 32.0% in Cork when the donor spike originates from the main shoot or the fourth tiller. Significantly, anthers collected from spike of the second tiller enabled us to drastically increase the proportion of regenerated green plantlets, by 16% in Igri and 1800% in Cork.     

The anther-culture response of triticale line x tester progenies

Seven triticale cultivars (Ampiac, Aubrac, Trinidad, Ticino, Lamberto, Pronto and Prado) and their F1 hybrids obtained after crossing in a line x tester scheme were examined with respect to their androgenetic effectiveness. The embryo induction rate (number of embryos per 100 anthers), green plant regeneration rate (number of green plantlets per 100 embryos), plant yield (number of green and albino plantlets per 100 anthers) and green plant yield (number of green plantlets per 100 anthers) were assessed. The multivariate and univariate effects of general (GCA) and specific (SCA) combining abilities for the studied traits were estimated and tested. Significant differences between the genotypes were found for individual traits as well as for all the traits treated jointly. Hybrids generally showed a better response in anther culture than their parental genotypes. Heterosis effects were observed in some hybrids for embryo induction rate and green plant yield. GCA and SCA variances were significant and a dominance of the GCA over the SCA variation was found. Among the examined cultivars, Ticino and Pronto were characterised by positive and significant GCA for embryo induction and green plant yield, and these cultivars may be recommended for the improvement of anther culture responsiveness in triticale.     

用花药培养创建小麦加倍单倍体作图群体

用花药培养创建加倍单倍体作图群体是构建数量性状基因作图群体的有效途径,但通过花药培养生产小麦加倍单倍体的成功率较低。该文考查了小麦幼穗发育时期、低温处理幼穗及高温处理接种花药对花粉愈伤诱导率的影响。采用春播小麦做为花药供体,推迟花药接种时间,避免试管苗在低温条件下越夏,有利于早移栽,培育冬前壮苗,同时加强试管苗移栽后的田间管理,保证安全越冬,有效地提高了花药培养生产加倍单倍体的成功率。通过花药培养创建了一个具有１９１个个体的小麦加倍单倍体作图群体,该群体将用于小麦有关抗旱性状及产量性状基因的遗传作图     

<Emphasis Type="Italic">In vitro</Emphasis> studies to produce double haploid in <Emphasis Type="Italic">Indica</Emphasis> hybrid rice

The aim of this investigation was to improve in vitro the technique of production of double haploid in Indica hybrid rice by combining anther culture, hormone shock and doubling chromosome. It was discussed how to avoid somaclonal variation during culturing and to reduce the time of this process. The anthers of KDML 105 × SPR 1 (Indica × Indica) were cultured in Linsmaier and Skoog (LS) medium, which contained nutrients, growth regulators [(2,4,-dichlorophenoxy acetic acid (2,4-D) and naphthalene acetic acid (NAA)] and organic compounds, and then subcultured by inducing embryo-like structure (ELS) LS media. During 4 weeks used LS media supplemented with 10 μM KNO³ + 2 mg/L 2,4-D + 2 mg/L NAA + 20% coconut water + 1 mg/L of activated charcoal had induced high embryogenic frequent callus with length of 4–5 mm. The supplementation of 0.2 g/L colchicine and 100 μM 2,4-D was the most efficient in LS media. Over 70% of viable double haploid ELS were produced in 8 weeks and subcultured only twice compared with conventional anther which takes more than 12 weeks. This new technique can therefore be applied to rice in order in shorten time to produce higher number of double haploid plantlets.